KR102175138B1 - Chryseobacterium indologenes strain possessing antifungal activity against pathogenic bacteria Pseudomonas syringae and use thereof - Google Patents
Chryseobacterium indologenes strain possessing antifungal activity against pathogenic bacteria Pseudomonas syringae and use thereof Download PDFInfo
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Abstract
본 발명은 슈도모나스 시링게(Pseudomonas syringae)에 대하여 항균활성을 갖는 크라이세오박테리움 인돌로제네스(Chryseobacterium indologenes) 균주에 대한 것이다.
또한, 본 발명은 상기 균주, 이의 배양액 및 이들의 혼합물로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는 슈도모나스 시링게(Pseudomonas syringae) 방제용 조성물 내지 상기 조성물을 처리하는 단계를 포함하는 슈도모나스 시링게(Pseudomonas syringae) 방제 방법에 대한 것이다.
따라서, 크라이세오박테리움 인돌로제네스 균주는 식물, 특히 토마토의 잎점무늬병을 일으키는 슈도모나스 시링게 균주에 대하여 항균활성이 뛰어나므로 슈도모나스 시링게 방제용 조성물 내지 방제 방법에 효과적으로 사용될 수 있다. 특히 상기 효과는 본 발명의 크라이세오박테리움 인돌로제네스 PgBE177(수탁번호: KACC 92247P)에 의하여 가장 유의적으로 얻을 수 있다.The present invention relates to a strain of Chryseobacterium indologenes having antibacterial activity against Pseudomonas syringae .
In addition, the present invention is a Pseudomonas syringae ( Pseudomonas syringae ) containing any one or more selected from the group consisting of the strain, a culture solution thereof, and a mixture thereof. Pseudomonas syringae ) control method.
Therefore, the Cryseobacterium indologenes strain has excellent antibacterial activity against plants, particularly Pseudomonas shiringe strains that cause leaf spot disease of tomatoes, and thus can be effectively used in compositions or methods for controlling Pseudomonas shiringe. In particular, the above effect can be obtained most significantly by the Cryseobacterium indologenes PgBE177 (accession number: KACC 92247P) of the present invention.
Description
본 발명은 슈도모나스 시링게(Pseudomonas syringae)에 대하여 항균활성을 갖는 크라이세오박테리움 인돌로제네스(Chryseobacterium indologenes) 균주에 대한 것이다. The present invention relates to a strain of Chryseobacterium indologenes having antibacterial activity against Pseudomonas syringae .
또한, 본 발명은 상기 균주, 이의 배양액 및 이들의 혼합물로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는 슈도모나스 시링게(Pseudomonas syringae) 방제용 조성물 내지 상기 조성물을 처리하는 단계를 포함하는 슈도모나스 시링게(Pseudomonas syringae) 방제 방법에 대한 것이다.In addition, the present invention is Pseudomonas siringe ( Pseudomonas) containing any one or more selected from the group consisting of the strain, its culture medium and a mixture thereof syringae ) Controlling composition to Pseudomonas syringae comprising the step of treating the composition ( Pseudomonas syringae ) to a control method.
반점세균병(bacterial speck disease)은 세균의 기생에 의해 발생하는 작물병으로, 잎, 줄기 또는 과실 등에 조그마한 담갈색, 또는 갈색반점이 생기는 증상이 나타난다. 잎의 경우에는 잎맥에 한하여 모난 병반이 생기는 경우도 있으며, 심할 때에는 병반이 융합하여 잎이 시든다. 사상균의 기생에 의한 반점병과는 달리, 병반에서 곰팡이의 발생은 찾아볼 수 없으나, 습도가 높을 때 병반 주위에 균니(세균 덩어리)가 고름 모양으로 스며나온다. 병원균은 작물의 종류에 따라 다른데, 슈도모나스(Pseudomonas)속 세균에 의한 것은 오이, 콩, 카네이션 등 18종의 작물에 발생며, 특히 하우스재배 작물에 피해가 매우 심하다. Bacterial speck disease is a crop disease caused by a parasitization of bacteria. A small pale brown or brown spot appears on leaves, stems, or fruits. In the case of leaves, angular lesions may occur only in leaf veins, and in severe cases, lesions fuse and leaves wither. Unlike spot diseases caused by filamentous fungi, fungi cannot be found in the lesion, but when the humidity is high, bacteria (bacterial mass) ooze around the lesion in the form of pus. Pathogens vary depending on the type of crop, but Pseudomonas ( Pseudomonas ) caused by bacteria occurs in 18 kinds of crops such as cucumbers, beans, carnations, especially house cultivation crops are very damaging.
슈도모나스 시링게(Pseudomonas syringae pv. tomato; Pst) 균주에 의한 세균성점무늬병(bacterial speck disease)은 온실 재배에서 토마토의 생산량을 감소키는 것으로 알려져 있다. 특히, 슈도모나스 시링게 pv. 토마토 DC3000(P.syringae pv. tomato DC3000), 슈도모나스 시링게 pv. 토마토 LMG5093(P.syringae pv. tomato LMG5093) 그리고 슈도모나스 시링게 pv. 토마토 OH314(P.syringae pv. tomato OH314)는 슈도모나스 시링게 pv. 토마토(P.syringae pv. tomato)의 일종으로, 대표적인 토마토 반점세균병의 병원균으로 알려져 있다. 화학약품의 살포가 상기 슈도모나스 시링개 균주에 의한 세균성 점무늬 병에 효과적이라는 것이 연구된 바 있지만, 이러한 농약의 사용은 환경 친화적이지 않고 병원균의 내성 증가시키며 유용한 생물에게 해롭다는 단점이 있다.Pseudomonas syringae ( Pseudomonas syringae pv. tomato ; Pst) strain caused by bacterial speck disease (bacterial speck disease) is known to reduce the production of tomatoes in greenhouse cultivation. In particular, Pseudomonas Shiringe pv. Tomato DC3000 (P. syringae pv. tomato DC3000), Pseudomonas shiringe pv. Tomato LMG5093 (P. syringae pv. tomato LMG5093) and Pseudomonas shiringe pv. Tomato OH314 (P. syringae pv. tomato OH314) is Pseudomonas Shiringe pv. It is a kind of tomato (P. syringae pv. tomato ), and is known as a representative pathogen of tomato spot bacterial disease. Although it has been studied that spraying of chemicals is effective against bacterial spotting disease caused by the Pseudomonas Shiringe strain, the use of such pesticides is not environmentally friendly, increases the resistance of pathogens, and is harmful to useful organisms.
내생 박테리아(endophytic bacteria)는 질병 증상을 일으키지 않고 식물체에 서식하고 상호 작용한다. 그들은 식물 종에 편재하고 다양한 식물 조직에 서식하며 식물 성장, 식물 병원체 억제, 식물 영양 및 항생제 생산의 향상과 같은 유익한 효과를 가질 수 있다. 숙주 식물에 의해 제공되는 안정된 서식지 때문에, 내생 박테리아는 영양소에 대한 경쟁이 적고 다양한 외부 스트레스로부터 보호된다. 따라서 이 박테리아는 식물 외부에 존재하는 엽권(phyllosphere)균 또는 근권(rhizosphere)균보다 더 일관된 효과를 발휘할 수 있다.Endophytic bacteria inhabit and interact with plants without causing disease symptoms. They are ubiquitous in plant species, inhabit a variety of plant tissues, and can have beneficial effects such as plant growth, suppression of plant pathogens, improvement of plant nutrition and antibiotic production. Because of the stable habitat provided by the host plant, endogenous bacteria have little competition for nutrients and are protected from various external stresses. Thus, these bacteria can exert a more consistent effect than phyllosphere or rhizosphere bacteria that exist outside the plant.
대한민국 공개특허 제10-2009-0058106호는 메틸로박테리움 오라이자 씨비엠비 20 균주를 포함하는 토마토의 세균성 점무늬병 방제용 미생물 제제에 대한 것으로서, 상기 균주는 슈도모나스 시링게 균주의 활성을 억제하여 토마토의 세균성 점무늬병을 효과적으로 억제할 수 있음을 개시하고 있다. 그러나, 인삼 내생균 중 본 발명의 크라이세오박테리움 인돌로제네스가 슈도모나스 시링게 균주 내지 토마토 세균성 점무늬병에 대하여 방제효과를 갖는지 여부에 대한 연구 내지 기재는 개시된 바 없다. Republic of Korea Patent Publication No. 10-2009-0058106 relates to a microbial preparation for controlling bacterial spotting disease of tomatoes, including 20 strains of Methylobacterium oriza CBMB, wherein the strain inhibits the activity of the Pseudomonas Shiringe strain. It is disclosed that it is possible to effectively suppress bacterial spotty disease. However, among ginseng endogenous bacteria, studies or descriptions on whether the Cryseobacterium indologenes of the present invention has a control effect against Pseudomonas shiringe strains or tomato bacterial spots have not been disclosed.
이에, 본 발명자들은 슈도모나스 시링게(Pseudomonas syringae)에 대한 방제제로서 사용할 수 있는 생물학적 방제제를 제공하기 위해 예의 노력한 결과, 인삼 내생균주 중 신규한 크라이세오박테리움 인돌로제네스(Chryseobacterium indologenes) 균주로서 크라이세오박테리움 인돌로제네스 PgBE177 균주가 슈도모나스 시링게에 대하여 항균활성을 나타냄을 확인하고 본 발명을 완성하였다. Accordingly, the present inventors have made diligent efforts to provide a biological control agent that can be used as a control agent for Pseudomonas syringae , as a result of a novel Chryseobacterium indologenes strain among ginseng endogenous strains. It was confirmed that the Cryseobacterium indologenes PgBE177 strain exhibited antimicrobial activity against Pseudomonas syringe, and the present invention was completed.
따라서, 본 발명의 목적은 슈도모나스 시링게(Pseudomonas syringae)에 대하여 항균활성을 갖는 크라이세오박테리움 인돌로제네스(Chryseobacterium indologenes) 균주를 제공하는 것이다. Accordingly, an object of the present invention is to provide a Chryseobacterium indologenes strain having antibacterial activity against Pseudomonas syringae .
본 발명의 또 다른 목적은, 상기 균주, 이의 배양액 및 이들의 혼합물로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는 슈도모나스 시링게(Pseudomonas syringae) 방제용 조성물 내지 이를 처리하는 단계를 포함하는 슈도모나스 시링게(Pseudomonas syringae) 방제 방법을 제공하는 것이다.Another object of the present invention is a composition for controlling Pseudomonas syringae comprising any one or more selected from the group consisting of the strain, a culture solution thereof, and a mixture thereof, or a Pseudomonas syringe comprising the step of treating the same ( Pseudomonas syringae ) to provide a control method.
상기 목적을 달성하기 위해, 본 발명은 슈도모나스 시링게(Pseudomonas syringae)에 대하여 항균활성을 갖는 크라이세오박테리움 인돌로제네스(Chryseobacterium indologenes) 균주를 제공한다. In order to achieve the above object, the present invention provides a Chryseobacterium indologenes strain having antimicrobial activity against Pseudomonas syringae .
본 발명의 바람직한 일실시예에 따르면, 상기 슈도모나스 시링게는 슈도모나스 시링게 pv. 토마토(Pseudomonas syringae pv. tomato)인 것일 수 있다. According to a preferred embodiment of the present invention, the Pseudomonas Shiringe is Pseudomonas Shiringe pv. It may be a tomato ( Pseudomonas syringae pv. tomato ).
본 발명의 바람직한 일실시예에 따르면, 상기 슈도모나스 시링게 pv. 토마토(Pseudomonas syringae pv. tomato)는 슈도모나스 시링게 pv. 토마토 DC3000(P.syringae pv. tomato DC3000), 슈도모나스 시링게 pv. 토마토 LMG5093(P.syringae pv. tomato LMG5093) 및 슈도모나스 시링게 pv. 토마토 OH314(P.syringae pv. tomato OH314)로 이루어진 군에서 선택되는 어느 하나 이상인 것일 수 있다. According to a preferred embodiment of the present invention, the Pseudomonas Shiringe pv. Tomato ( Pseudomonas syringae pv. tomato ) is Pseudomonas Shiringe pv. Tomato DC3000 (P. syringae pv. tomato DC3000), Pseudomonas shiringe pv. Tomato LMG5093 (P. syringae pv. tomato LMG5093) and Pseudomonas shiringe pv. Tomato OH314 (P. syringae pv. tomato OH314) may be any one or more selected from the group consisting of.
본 발명의 바람직한 일실시예에 따르면, 상기 슈도모나스 시링게는 식물에 세균성 잎점무늬병(bacterial speck disease)을 일으키는 것일 수 있다. According to a preferred embodiment of the present invention, the Pseudomonas syringe may cause bacterial speck disease in plants.
본 발명의 바람직한 일실시예에 따르면, 상기 식물은 고추, 애기장대 및 토마토로 이루어진 군에서 선택되는 어느 하나 이상인 것일 수 있다. According to a preferred embodiment of the present invention, the plant may be any one or more selected from the group consisting of peppers, Arabidopsis and tomatoes.
본 발명의 바람직한 일실시예에 따르면, 상기 균주는 서열번호 1의 염기서열을 포함하는 것일 수 있다. According to a preferred embodiment of the present invention, the strain may include the nucleotide sequence of SEQ ID NO: 1.
본 발명의 바람직한 일실시예에 따르면, 상기 균주는 인삼의 뿌리, 줄기, 잎, 꽃, 꽃 줄기 및 전초로 이루어진 군에서 선택되는 어느 하나 이상의 부위에서 내생하는 균주인 것일 수 있다. According to a preferred embodiment of the present invention, the strain may be a strain endogenous at any one or more sites selected from the group consisting of roots, stems, leaves, flowers, flower stems, and outposts of ginseng.
본 발명의 바람직한 일실시예에 따르면, 상기 균주는 수탁번호 KACC 92247P의 크라이세오박테리움 인돌로제네스 PgBE177 균주인 것일 수 있다. According to a preferred embodiment of the present invention, the strain may be a Cryseobacterium indologenes PgBE177 strain of accession number KACC 92247P.
본 발명은 또한, 상기 균주, 이의 배양액 및 이들의 혼합물로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는 슈도모나스 시링게(Pseudomonas syringae) 방제용 조성물을 제공한다. The present invention also provides a composition for controlling Pseudomonas syringae ( Pseudomonas syringae ) comprising any one or more selected from the group consisting of the strain, a culture solution thereof, and a mixture thereof.
본 발명의 바람직한 일실시예에 따르면, 상기 식물은 고추, 애기장대 및 토마토로 이루어진 군에서 선택되는 어느 하나 이상인 것일 수 있다. According to a preferred embodiment of the present invention, the plant may be any one or more selected from the group consisting of peppers, Arabidopsis and tomatoes.
본 발명은 또한, 상기 방제용 조성물을 처리하는 단계를 포함하는 슈도모나스 시링게(Pseudomonas syringae) 방제 방법을 제공한다. The present invention also provides a Pseudomonas syringae ( Pseudomonas syringae ) control method comprising the step of treating the control composition.
본 발명의 바람직한 일실시예에 따르면, 상기 식물은 고추, 애기장대 및 토마토로 이루어진 군에서 선택되는 어느 하나 이상인 것일 수 있다.According to a preferred embodiment of the present invention, the plant may be any one or more selected from the group consisting of peppers, Arabidopsis and tomatoes.
크라이세오박테리움 인돌로제네스 균주는 식물, 특히 토마토의 잎점무늬병을 일으키는 슈도모나스 시링게 균주에 대하여 항균활성이 뛰어나므로 슈도모나스 시링게 방제용 조성물 내지 방제 방법에 효과적으로 사용될 수 있다. 특히 상기 효과는 본 발명의 크라이세오박테리움 인돌로제네스 PgBE177(수탁번호: KACC 92247P)에 의하여 가장 유의적으로 얻을 수 있다.Since the Cryseobacterium indologenes strain has excellent antibacterial activity against plants, particularly Pseudomonas shiringe strains that cause leaf spot disease of tomatoes, it can be effectively used in compositions or methods for controlling Pseudomonas shiringe. In particular, the above effect can be obtained most significantly by the Cryseobacterium indologenes PgBE177 (accession number: KACC 92247P) of the present invention.
도 1은 본 발명의 크라이세오박테리움 인돌로제네스 PgBE177 균주가 슈도모나스 시링게 pv. 토마토 DC3000 균주에 항균활성을 가짐을 나타낸다.
도 2는 본 발명의 크라이세오박테리움 인돌로제네스 PgBE177 균주의 유전체지도를 나타낸다.1 is a Cryseobacterium indologenes PgBE177 strain of the present invention is Pseudomonas Shiringe pv. It shows that it has antibacterial activity in tomato DC3000 strain.
Figure 2 shows the genomic map of the Cryseobacterium indologenes PgBE177 strain of the present invention.
이하, 본 발명을 보다 상세히 설명한다. Hereinafter, the present invention will be described in more detail.
상술한 바와 같이, 종래 기술에서는 식물, 특히 토마토의 잎점무늬병을 방제하기 위하여 주로 화학적 방제방법을 이용하고 있었으나 효과가 지속적이지 않고 내성균 발생, 독성으로 인해 토양 생태계 파괴 내지 식물에 독성이 잔류하여 결과적으로 이를 섭취하는 포유류에 독성을 띌 가능성 등의 문제점을 가졌다. 생물적 방제방법도 연구가 되고 있긴 하였으나 그 효과가 미미하였다. As described above, in the prior art, chemical control methods were mainly used to control leaf spots of plants, especially tomatoes, but the effect was not sustained, and the soil ecosystem was destroyed due to the generation of resistant bacteria and toxicity, or toxicity remained in the plants. It has problems such as the possibility of being toxic to mammals that consume it. Although biological control methods were also being studied, their effects were insignificant.
본 발명에 따른 크라이세오박테리움 인돌로제네스(Chryseobacterium indologenes) 균주, 특히 크라이세오박테리움 인돌로제네스 PgBE177는 인삼 내생균주로서 친환경적이며, 슈도모나스 시링게(Pseudomonas syringae)에 대한 항균효과가 유의적이므로 슈도모나스 시링게 방제용 조성물 내지 방제 방법에 효과적으로 이용될 수 있다. Chryseobacterium indologenes strain according to the present invention, in particular, Cryseobacterium indologenes PgBE177 is an eco-friendly ginseng endogenous strain, and Pseudomonas syringae has a significant antibacterial effect against Pseudomonas siringae . It can be effectively used in the composition or control method for controlling Ringae.
따라서, 본 발명은 슈도모나스 시링게(Pseudomonas syringae)에 대하여 항균활성을 갖는 크라이세오박테리움 인돌로제네스(Chryseobacterium indologenes) 균주를 제공할 수 있다. Accordingly, the present invention can provide a Chryseobacterium indologenes strain having antimicrobial activity against Pseudomonas syringae .
본 발명의 상기 슈도모나스 시링게는 Pseudomonas syringae pv. actinidiae, Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. lachrymans, Pseudomonas syringae pv. tabaci, Pseudomonas syringae pv. glycines 및 Pseudomonas syringae pv. tomato 로 이루어진 군에서 선택되는 어느 하나 이상인 것이 바람직하며, 더욱 바람직하게는 슈도모나스 시링게는 슈도모나스 시링게 pv. 토마토(Pseudomonas syringae pv. tomato) 인 것이 바람직하다. The Pseudomonas syringae of the present invention is Pseudomonas syringae pv. actinidiae , Pseudomonas syringae pv. syringae , Pseudomonas syringae pv. lachrymans , Pseudomonas syringae pv. tabaci , Pseudomonas syringae pv. glycines and Pseudomonas syringae pv. It is preferably any one or more selected from the group consisting of tomato , more preferably Pseudomonas Shiringe is Pseudomonas Shiringe pv. Tomato ( Pseudomonas syringae pv. tomato ) is preferred.
본 발명의 상기 슈도모나스 시링게 pv. 토마토(Pseudomonas syringae pv. tomato)는 슈도모나스 시링게 pv. 토마토 DC3000(P.syringae pv. tomato DC3000), 슈도모나스 시링게 pv. 토마토 LMG5093(P.syringae pv. tomato LMG5093) 및 슈도모나스 시링게 pv. 토마토 OH314(P.syringae pv. tomato OH314)로 이루어진 군에서 선택되는 어느 하나 이상인 것이 바람직하며, 더욱 바람직하게는 Pseudomonas syringae pv. tomato DC3000 인 것이 바람직하다. The Pseudomonas Shiringe pv. Tomato ( Pseudomonas syringae pv. tomato ) is Pseudomonas syringae pv. Tomato DC3000 (P. syringae pv. tomato DC3000), Pseudomonas shiringe pv. Tomato LMG5093 (P. syringae pv. tomato LMG5093) and Pseudomonas shiringe pv. Tomato OH314 (P. syringae pv. tomato OH314) is preferably any one or more selected from the group consisting of, more preferably Pseudomonas syringae pv. Tomato DC3000 is preferred.
상기 슈도모나스 시링게 pv. 토마토 DC3000(Pseudomonas syringae pv. tomato DC3000)은 고추, 애기장대 또는 토마토 등의 작물에 세균성 잎점무늬병을 일으키는 병원균으로 알려져 있다. The Pseudomonas Siringe pv. Tomato DC3000 ( Pseudomonas syringae pv. tomato DC3000) is known as a pathogen that causes bacterial leaf spot disease in crops such as pepper, Arabidopsis or tomato.
본 발명의 상기 슈도모나스 시링게는 식물에 세균성 잎점무늬병(bacterial speck disease)을 일으키는 것이 바람직하나 이에 한정되지 않는다. The Pseudomonas syringe of the present invention is preferably to cause bacterial speck disease in plants, but is not limited thereto.
본 발명의 상기 식물은 고추, 애기장대 및 토마토로 이루어진 군에서 선택되는 어느 하나 이상인 것이 바람직하나, 가장 바람직하게는 토마토인 것이 가장 바람직하다. The plant of the present invention is preferably any one or more selected from the group consisting of peppers, Arabidopsis, and tomatoes, most preferably tomatoes.
본 발명의 상기 균주는 서열번호 1의 염기서열을 포함하는 것이 바람직하며, 더욱 바람직하게는 수탁번호 KACC 92247P의 크라이세오박테리움 인돌로제네스 PgBE177 균주인 것이 더욱 바람직하다. It is preferable that the strain of the present invention contains the nucleotide sequence of SEQ ID NO: 1, and more preferably, it is more preferably a Cryseobacterium indologenes PgBE177 strain of accession number KACC 92247P.
본 발명의 상기 균주는 인삼의 뿌리, 줄기, 잎, 꽃, 꽃 줄기 및 전초로 이루어진 군에서 선택되는 어느 하나 이상의 부위에서 내생하는 균주인 것이 바람직하나, 더욱 바람직하게는 인삼의 뿌리인 것이 바람직하다. 상기 인삼은 미국삼(Panax quinquefolius)인 것이 바람직하나 이에 한정되지 않는다. The strain of the present invention is preferably a strain endogenous at any one or more sites selected from the group consisting of ginseng roots, stems, leaves, flowers, flower stems and outposts, more preferably ginseng roots. . The ginseng is preferably American ginseng ( Panax quinquefolius ), but is not limited thereto.
본 발명은 또한, 상기 균주, 이의 배양액 및 이들의 혼합물로 이루어진 군에서 선택되는 어느 하나 이상을 포함하는 슈도모나스 시링게(Pseudomonas syringae) 방제용 조성물을 제공할 수 있다. The present invention can also provide a composition for controlling Pseudomonas syringae ( Pseudomonas syringae ) comprising any one or more selected from the group consisting of the strain, its culture medium, and a mixture thereof.
상기 배양액은 미생물 생장에 적절한 배지에 일정 농도의 포자를 접종하여 배양기에서 일정 기간 동안 미생물이 생장하도록 유도한 후 수득된, 균체, 균체가 생장하면서 분비한 물질 및 생장에 필요한 영양성분이 혼합된 액으로서, 불순물을 걸러내는 과정을 거쳐 제조되는 것을 의미한다. 구체적으로, 본 발명의 크라이세오박테리움 인돌로제네스 균주를 LB(Luria Bertani) 배지, NB(nutrient broth) 배지, BHI(brain heart infusion) 배지, KB(King's medium B) 또는 TSB(Tryptic Soy Broth) 배지로 구성된 군에서 선택되는 어느 하나 이상의 배지에서 배양하여 제조되는 것을 의미하는 것이 바람직하나 이에 한정되지 않는다. The culture solution is obtained after inoculating a certain concentration of spores in a medium suitable for growth of microorganisms to induce the growth of microorganisms for a certain period in an incubator, and a mixture of the cells, substances secreted while the cells are growing, and nutrients necessary for growth As, it means that it is manufactured through a process of filtering out impurities. Specifically, the Cryseobacterium indologenes strain of the present invention was used as LB (Luria Bertani) medium, NB (nutrient broth) medium, BHI (brain heart infusion) medium, KB (King's medium B) or TSB (Tryptic Soy Broth) It is preferable to mean that it is produced by culturing in any one or more media selected from the group consisting of media, but is not limited thereto.
본 발명의 상기 식물은 고추, 애기장대 및 토마토로 이루어진 군에서 선택되는 어느 하나 이상인 것이 바람직하나 이에 한정되지 않는다. The plant of the present invention is preferably any one or more selected from the group consisting of pepper, Arabidopsis and tomato, but is not limited thereto.
상기 슈도모나스 시링게(Pseudomonas syringae)는 본 발명의 크라이세오박테리움 인돌로제네스(Chryseobacterium indologenes) 균주가 항균활성을 갖는 균주와 동일하므로 설명은 그 기재로 대신한다. The Pseudomonas syringae ( Pseudomonas syringae ) is the same as the strain of the Chryseobacterium indologenes ( Chryseobacterium indologenes ) strain of the present invention having antibacterial activity, so the description will be replaced with the description.
상기 방제용 조성물은 식물 병원균으로 인한 병의 예방, 감소 또는 근절을 위해 사용되는 통상의 방제제라면 특별히 제한하지 않으며, 상기 예방 또는 감소는 상기 조성물이 비처리된 기주식물에 대하여 통계적으로 현저한 차이가 있음을 의미한다. 바람직하게는 미생물농약, 종자코팅제, 슈도모나스 시링게 방제용 미생물농약, 작물재배용 무살균 배지제조를 위한 첨가제, 바이오상토, 미생물영양제, 토양개량제, 퇴비부숙제, 엽면살포제 또는 관주살포제로 이루어진 군에서 선택되는 어느 하나 이상일 수 있다. 특히, 토마토 잎점무늬병의 병원균인 슈도모나스 시링게pv. 토마토(Pseudomonas syringae pv. tomato)의 성장억제에 탁월한 효과가 있어, 토마토의 잎점무늬병 방제용 미생물 농약, 토마토재배용 무살균 배지제조를 위한 첨가제, 바이오상토의 제조에도 활용될 수 있다. 상기 미생물농약은 본 발명의 크라이세오박테리움 인돌로제네스 균주를 포함하여 통상의 방법으로 제조된 것이라면 특별히 제한하지 않는다. 예를 들면, 크라이세오박테리움 인돌로제네스 균주 및 미생물 담체인 바이오 매트릭스를 포함하는 것일 수 있으며, 상기 바이오 매트릭스는 식물자원 고분자 재료, 미생물 기원 생체 고분자 재료, 영양제, 보조 재료 및 첨가제를 포함할 수 있다. 또한, 상기 식물자원 고분자 재료는 감자가루, 콩가루, 전분가루 등을 포함할 수 있으며, 상기 미생물기원 생체 고분자는 뮤코폴리사카라이드, 폴리-베타하이드록시-알카노에이트 및 응집제를 포함할 수 있다. 나아가, 상기 영양제는 포도당, 펩톤 및 무기염류를 포함할 수 있으며, 상기 보조 재료는 질석, 왕겨 태운가루 등을 포함할 수 있고, 상기 첨가제는 증량제, UV 차단제, 계면활성제를 포함할 수 있다. 상기 미생물농약은 항진균 미생물농약, 종자 코팅제, 미생물영양제, 토양개량제, 퇴비부숙제, 엽면살포제 또는 관주살포제 등으로 사용될 수 있다.The composition for control is not particularly limited as long as it is a conventional control agent used for the prevention, reduction or eradication of diseases caused by plant pathogens, and the prevention or reduction has a statistically significant difference with respect to the host plant in which the composition is untreated. Means there is. Preferably, it is selected from the group consisting of microbial pesticides, seed coating agents, microbial pesticides for controlling Pseudomonas shiringe, additives for producing a non-sterile medium for crop cultivation, biotop soil, microbial nutrients, soil improvers, composting agents, foliar sprayers or irrigation sprayers It may be any one or more. In particular, Pseudomonas Shiringe pv, a pathogen of tomato leaf spot disease. Tomato ( Pseudomonas syringae pv. tomato ) has an excellent effect on growth inhibition, so it can be used for microbial pesticides for controlling leaf spots on tomatoes, additives for the production of non-sterile medium for tomato cultivation, and also for the production of biotope. The microbial pesticide is not particularly limited as long as it is prepared by a conventional method, including the Cryseobacterium indologenes strain of the present invention. For example, it may include a Cryseobacterium indologenes strain and a bio-matrix that is a microbial carrier, and the bio-matrix may include a plant resource polymer material, a microbial-derived biopolymer material, a nutrient, an auxiliary material, and an additive. have. In addition, the plant resource polymer material may include potato powder, soybean powder, starch powder, and the like, and the microbial-based biopolymer may include mucopolysaccharide, poly-betahydroxy-alkanoate, and a coagulant. Further, the nutrient may include glucose, peptone and inorganic salts, and the auxiliary material may include vermiculite, rice husk burned powder, and the like, and the additive may include an extender, a UV blocker, and a surfactant. The microbial pesticide may be used as an antifungal microbial pesticide, a seed coating agent, a microbial nutrient agent, a soil conditioner, a composting agent, a foliar spray agent or a drench spray agent.
본 발명은 또한, 상기 방제용 조성물을 처리하는 단계를 포함하는 슈도모나스 시링게(Pseudomonas syringae) 방제 방법을 제공할 수 있다. The present invention may also provide a Pseudomonas syringae ( Pseudomonas syringae ) control method comprising the step of treating the control composition.
본 발명의 상기 식물은 고추, 애기장대 및 토마토로 이루어진 군에서 선택되는 어느 하나 이상인 것이 바람직하나 이에 한정되지 않는다. The plant of the present invention is preferably any one or more selected from the group consisting of pepper, Arabidopsis and tomato, but is not limited thereto.
본 발명의 상기 방제 방법은 상기 방제용 조성물을 토양, 식물 또는 식물 종자에 처리함으로써 토마토 잎점무늬병을 방제하는 방법일 수 있다. 이때, 처리방법에는 일반적으로 행하고 있는 방법, 즉 살포(예를 들면 분무, 미스팅, 아토마이징, 분말 살포, 과립 살포, 수면시용, 상시용 등), 토양시용(예를 들면 혼입, 관주 등), 표면사용(예를 들면 도포, 도말법, 피복 등), 침지, 독이, 훈연 시용 등에 의해 행할 수 있다. 그 사용량은, 그 제형, 피해상황, 적용방법, 적용장소 등에 따라 적절히 결정할 수 있다. 본 발명에서, 상기 방법에 따라 처리되는 제제에 함유된 미생물의 유효량은 경작지 면적(㎡) 당 1 내지 1×10100의 미생물 수로 포함될 수 있다. 또한, 상기 방법 중 살포에 의해 처리되는 제제에 함유된 미생물의 유효량은 ㎖당 1 내지 1×10100의 미생물 농도로 포함될 수 있으며, 침지에 의해 처리되는 조성물에 함유된 미생물의 유효량은 ㎖당 1 내지 1×10100의 미생물 농도로 포함될 수 있다.The control method of the present invention may be a method of controlling tomato leaf spots by treating the control composition to soil, plants, or plant seeds. At this time, treatment methods are generally used, namely, spraying (e.g. spraying, misting, atomizing, powder spraying, granule spraying, water surface application, regular use, etc.), soil application (e.g. mixing, irrigation, etc.) , Surface use (for example, coating, smearing, coating, etc.), immersion, poisoning, smoking, etc. can be performed. The amount of use can be appropriately determined depending on the formulation, damage situation, application method, application location, and the like. In the present invention, the effective amount of microorganisms contained in the formulation treated according to the above method may be included in the number of microorganisms of 1 to 1 × 10 100 per cultivated land area (m 2 ). In addition, the effective amount of microorganisms contained in the formulation treated by spraying among the above methods may be included in a concentration of microorganisms of 1 to 1×10 100 per ml, and the effective amount of microorganisms contained in the composition treated by immersion is 1 per ml. It may be included in a microbial concentration of 1 × 10 100 .
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다. Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.
내생세균 분리 및 동정 Isolation and identification of endogenous bacteria
국립원예특작과학원 인삼특작부 인삼포장에서 4년생 인삼(Panax quinquefolius)의 뿌리를 확보하였다. 상기 뿌리 샘플은 수돗물로 세척하여 뿌리 표면의 흙과 이물질을 제거한 후, 12% 락스와 70% 에탄올로 소독한 다음 멸균수로 헹구었다. 마쇄한 샘플 500 mg을 멸균수 1 ㎖과 혼합하여 100 ㎕를 취한 후 LB(Luria-Bertani) 플레이트에 도말하였다. 상온에서 2일 배양 후 나타나는 단일 콜로니를 계대배양한 후 16S rRNA 유전자 염기서열 분석을 진행하였다. 확보한 염기서열은 NCBI의 BLAST 검색을 통해 유사종을 확인하였다. National Horticultural Research Institute teukjak ginseng teukjak Part 4 - year - old ginseng ginseng packaging (Panax quinquefolius ) roots were secured. The root sample was washed with tap water to remove soil and foreign substances on the root surface, sterilized with 12% Lax and 70% ethanol, and then rinsed with sterile water. 500 mg of the ground sample was mixed with 1 ml of sterilized water to take 100 µl and then spread on an LB (Luria-Bertani) plate. A single colony that appeared after 2 days incubation at room temperature was subcultured and then 16S rRNA gene sequence analysis was performed. The obtained nucleotide sequence was identified as similar species through BLAST search of NCBI.
그 결과, 염기서열을 확인한 균주 중 PgBE177은 크라이세오박테리움 인돌로제네스(Chryseobacterium indologenes) 종인 것으로 확인하였다. 이는 GenBank에 수탁번호 MH211268로 기탁하였다. As a result, it was confirmed that PgBE177 among the strains whose base sequence was confirmed is a species of Chryseobacterium indologenes . It was deposited with GenBank under accession number MH211268.
식물 병원균 억제 활성 기내 검정Plant pathogen inhibition activity in-flight assay
분리 균주의 유용성을 알아보기 위해 내생세균 균주를 대상으로 식물 병원균에 대한 항균활성을 기내 검정하였다. In order to find out the usefulness of the isolated strain, the antibacterial activity against plant pathogens was tested in-flight targeting the endogenous bacterial strain.
대상 병원균은 슈도모나스 시링게 pv. 토마토 DC3000(Pseudomonas syringae pv. tomato DC3000)을 사용하였다. 병원균은 GFP 단백질이 태깅(tagging)된 것으로써 병원균 생장시 형광의 세기가 세지고, 억제시 형광이 약해지는 성질을 이용하여 병원균의 생장을 모니터링 하고자 사용되었다. PgBE177과 병원균을 28℃, 200 rpm 조건으로 20 시간 진탕 배양한 후, 각각의 배양액의 농도를 OD600 = 0.1로 맞추고 1:1로 섞은 다음 LB(Luria-Bertani) 플레이트에 떨어뜨렸다. 1~2일 후 병원균의 형광을 대조구와 비교하여 억제 활성을 확인하였다. The target pathogen was Pseudomonas Shiringe pv. Tomato DC3000 ( Pseudomonas syringae pv. tomato DC3000) was used. Pathogens were tagged with GFP protein, and were used to monitor the growth of pathogens by using the properties that the fluorescence intensity increases when pathogens grow and the fluorescence decreases when suppression. PgBE177 and pathogens were incubated with shaking at 28°C and 200 rpm for 20 hours, and then the concentration of each culture solution was adjusted to OD 600 = 0.1, mixed at 1:1, and then dropped on an LB (Luria-Bertani) plate. After 1-2 days, the inhibitory activity was confirmed by comparing the fluorescence of the pathogen with the control.
그 결과, [도 1]에 나타나는 바와 같이 본 발명의 PgBE177은 상기 병원균에 대하여 억제 활성을 나타냈다. As a result, as shown in [Fig. 1], the PgBE177 of the present invention exhibited inhibitory activity against the pathogen.
PgBE177의 전체염기서열분석Total base sequence analysis of PgBE177
본 발명의 PgBE177의 기능적 중요성을 밝히기 위해 PacBio RS II 시스템(Chunlab, Inc.)을 이용하여 전체염기서열을 분석하고자 하였다. In order to clarify the functional importance of PgBE177 of the present invention, the total base sequence was analyzed using the PacBio RS II system (Chunlab, Inc.).
구체적으로, 게놈 DNA 추출을 위해 박테리아 세포를 65 ℃에서 30 분 동안 Chelex 100 레진(카탈로그 번호 143-2832, Bio-Rad, CA, USA) 및 프로테이나제 K와 함께 항온 처리한 다음 100 ℃에서 10 분간 끓였다. 10,000X으로 10분 동안 원심분리하여 세균 게놈 DNA를 함유하는 상등액을 수득하였다. 샘플 준비는 제조사의 프로토콜에 따라 SMRTbell 템플릿 준비 키트(Pacific Biosciences, Menlo Park, CA, USA)를 사용했다. BluePippin size selection system을 사용하여 20-kb DNA 템플릿을 선택했다. DNA 크기 선택 후, 라이브러리는 8-well 단일 분자 실시간(SMRT) 셀 (버전 3)에서 P6-C4 화학(Pacific Biosciences)이 있는 PacBio RS II 기기를 사용하여 시퀀싱되었다. 어셈블리 프로세스는 HGAP2(Hierarchical Genome Assembly Process 2) 파이프라인을 사용하여 PacBio SMRT Analysis 2.3.0으로 수행되었다. Specifically, for genomic DNA extraction, bacterial cells were incubated at 65° C. for 30 minutes with Chelex 100 resin (Cat. No. 143-2832, Bio-Rad, CA, USA) and proteinase K, and then at 100° C. Boiled for 10 minutes. Centrifugation at 10,000X for 10 minutes to obtain a supernatant containing bacterial genomic DNA. For sample preparation, SMRTbell template preparation kit (Pacific Biosciences, Menlo Park, CA, USA) was used according to the manufacturer's protocol. 20-kb DNA templates were selected using the BluePippin size selection system. After DNA size selection, the library was sequenced using a PacBio RS II instrument with P6-C4 chemistry (Pacific Biosciences) in 8-well single molecule real-time (SMRT) cells (version 3). The assembly process was performed with PacBio SMRT Analysis 2.3.0 using the Hierarchical Genome Assembly Process 2 (HGAP2) pipeline.
추가적인 분석을 위해 RAST, antiSMASH 4.2.0 프로그램을 사용하여 게놈 시퀀스 분석을 수행하였다.For further analysis, genome sequence analysis was performed using RAST and antiSMASH 4.2.0 programs.
그 결과, 본 발명의 PgBE177은 1개의 contig로 구성되어 있으며 전체 게놈 크기 5,008,467 bp, GC 함량은 36.4% 이었으며, 커버리지의 깊이는 211.23×이었다. PgBE177은 9개의 유전자 클러스터를 보유하고 있었으며 식물 병원균 억제 활성에 관여하는 박테리오신 클러스터, 식물 생장 촉진에 관여하는 사이드로포어 관련 클러스터를 보유하고 있었다. As a result, the PgBE177 of the present invention was composed of one contig, the total genome size was 5,008,467 bp, the GC content was 36.4%, and the depth of coverage was 211.23×. PgBE177 had 9 gene clusters, a bacteriocin cluster involved in plant pathogen inhibitory activity, and a siderophore related cluster involved in plant growth promotion.
결과적으로, 본 발명의 PgBE177의 유전체 분석을 통하여 식물에 유용할 것으로 기대되는 형질을 확인할 수 있었으며, [도 2]는 본 발명의 크라이세오박테리움 인돌로제네스(Chryseobacterium indologenes) PgBE177의 유전체 지도를 나타낸다.As a result, through the genomic analysis of PgBE177 of the present invention, a trait expected to be useful in plants could be identified, and [Fig. 2] shows a genomic map of the Chryseobacterium indologenes PgBE177 of the present invention. .
[수탁기관] [Consignment institution]
기탁기관명 : 농업생명공학연구원Name of deposit institution: Institute of Agricultural Biotechnology
수탁번호 : KACC92247Accession number: KACC92247
수탁일자 : 20181023Consignment Date: 20181023
<110> REPUBLIC OF KOREA(MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> Chryseobacterium indologenes strain possessing antifungal activity against pathogenic bacteria Pseudomonas syringae and use thereof <130> 1065775 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1425 <212> DNA <213> Artificial Sequence <220> <223> PgBe177 16S ribosomal RNA gene <400> 1 gatgaacgct agcgggaggc ctaacacatg caagccgagc ggtagagatc tttcgggatc 60 ttgagagcgg cgtacgggtg cggaacacgt gtgcaacctg cctttatctg ggggatagcc 120 tttcgaaagg aagattaata ccccataata tttttgatgg catcattaga aattgaaaac 180 tccggtggat agagatgggc acgcgcaaga ttagatagtt ggtgaggtaa cggctcacca 240 agtctacgat ctttaggggg cctgagaggg tgatccccca cactggtact gagacacgga 300 ccagactcct acgggaggca gcagtgagga atattggaca atgggtgaga gcctgatcca 360 gccatcccgc gtgaaggacg acggccctat gggttgtaaa cttcttttgt atagggataa 420 acctactctc gtgagagtag ctgaaggtac tatacgaata agcaccggct aactccgtgc 480 cagcagccgc ggtaatacgg agggtgcaag cgttatccgg atttattggg tttaaagggt 540 ccgtaggcgg atctgtaagt cagtggtgaa atctcacagc ttaactgtga aactgccatt 600 gatactgcag gtcttgagtg ttattgaagt agctggaata agtagtgtag cggtgaaatg 660 catagatatt acttagaaca ccaattgcga aggcaggtta ctaagcaaca actgacgctg 720 atggacgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg 780 atgctaactc gtttttggtt tttcggaatc agagactaag cgaaagtgat aagttagcca 840 cctggggagt acgttcgcaa gaatgaaact caaaggaatt gacgggggcc cgcacaagcg 900 gtggattatg tggtttaatt cgatgatacg cgaggaacct taccaaggct taaatgggaa 960 atgacaggtt tagaaataga cttttcttcg gacatttttc aaggtgctgc atggttgtcg 1020 tcagctcgtg ccgtgaggtg ttaggttaag tcctgcaacg agcgcaaccc ctgtcactag 1080 ttgccatcat taagttgggg actctagtga gactgcctac gcaagtagag aggaaggtgg 1140 ggatgacgtc aaatcatcac ggcccttacg ccttgggcca cacacgtaat acaatggccg 1200 gtacagaggg cagctacaca gcgatgtgat gcaaatctcg aaagccggtc tcagttcgga 1260 ttggagtctg caactcgact ctatgaagct ggaatcgcta gtaatcgcgc atcagccatg 1320 gcgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcaagccatg gaagtctggg 1380 gtacctgaag tcggtgaccg taacaggagc tgcctagggt aaaac 1425 <110> REPUBLIC OF KOREA(MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Chryseobacterium indologenes strain possessing antifungal activity against pathogenic bacteria Pseudomonas syringae and use thereof <130> 1065775 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1425 <212> DNA <213> Artificial Sequence <220> <223> PgBe177 16S ribosomal RNA gene <400> 1 gatgaacgct agcgggaggc ctaacacatg caagccgagc ggtagagatc tttcgggatc 60 ttgagagcgg cgtacgggtg cggaacacgt gtgcaacctg cctttatctg ggggatagcc 120 tttcgaaagg aagattaata ccccataata tttttgatgg catcattaga aattgaaaac 180 tccggtggat agagatgggc acgcgcaaga ttagatagtt ggtgaggtaa cggctcacca 240 agtctacgat ctttaggggg cctgagaggg tgatccccca cactggtact gagacacgga 300 ccagactcct acgggaggca gcagtgagga atattggaca atgggtgaga gcctgatcca 360 gccatcccgc gtgaaggacg acggccctat gggttgtaaa cttcttttgt atagggataa 420 acctactctc gtgagagtag ctgaaggtac tatacgaata agcaccggct aactccgtgc 480 cagcagccgc ggtaatacgg agggtgcaag cgttatccgg atttattggg tttaaagggt 540 ccgtaggcgg atctgtaagt cagtggtgaa atctcacagc ttaactgtga aactgccatt 600 gatactgcag gtcttgagtg ttattgaagt agctggaata agtagtgtag cggtgaaatg 660 catagatatt acttagaaca ccaattgcga aggcaggtta ctaagcaaca actgacgctg 720 atggacgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg 780 atgctaactc gtttttggtt tttcggaatc agagactaag cgaaagtgat aagttagcca 840 cctggggagt acgttcgcaa gaatgaaact caaaggaatt gacgggggcc cgcacaagcg 900 gtggattatg tggtttaatt cgatgatacg cgaggaacct taccaaggct taaatgggaa 960 atgacaggtt tagaaataga cttttcttcg gacatttttc aaggtgctgc atggttgtcg 1020 tcagctcgtg ccgtgaggtg ttaggttaag tcctgcaacg agcgcaaccc ctgtcactag 1080 ttgccatcat taagttgggg actctagtga gactgcctac gcaagtagag aggaaggtgg 1140 ggatgacgtc aaatcatcac ggcccttacg ccttgggcca cacacgtaat acaatggccg 1200 gtacagaggg cagctacaca gcgatgtgat gcaaatctcg aaagccggtc tcagttcgga 1260 ttggagtctg caactcgact ctatgaagct ggaatcgcta gtaatcgcgc atcagccatg 1320 gcgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcaagccatg gaagtctggg 1380 gtacctgaag tcggtgaccg taacaggagc tgcctagggt aaaac 1425
Claims (12)
Pseudomonas syringae ( Pseudomonas syringae ) of the accession number KACC 92247P, which has antibacterial activity, Chryseobacterium indologenes PgBE177 strain.
The method of claim 1, wherein the Pseudomonas Shiringe is Pseudomonas Shiringe pv. Tomato ( Pseudomonas syringae pv. tomato ) of the accession number KACC 92247P, characterized in that the Chryseobacterium indologenes ( Chryseobacterium indologenes ) PgBE177 strain.
The method of claim 2, wherein the Pseudomonas Siringe pv. Tomato is Pseudomonas Shiringe pv. Tomato DC3000 (P. syringae pv. tomato DC3000), Pseudomonas shiringe pv. Tomato LMG5093 (P. syringae pv. tomato LMG5093) and Pseudomonas shiringe pv. Tomato OH314 (P. syringae pv. tomato OH314) Cryseobacterium indologenes ( Chryseobacterium indologenes ) PgBE177 strain of accession number KACC 92247P, characterized in that any one or more selected from the group consisting of.
The strain of claim 1, wherein the Pseudomonas syringe causes bacterial speck disease in plants. Chryseobacterium indologenes PgBE177 of accession number KACC 92247P.
According to claim 4, The plant is Chryseobacterium indologenes ( Chryseobacterium indologenes ) PgBE177 strain of accession number KACC 92247P, characterized in that any one or more selected from the group consisting of pepper, Arabidopsis and tomato.
The strain of claim 1, wherein the strain comprises the nucleotide sequence of SEQ ID NO: 1, Chryseobacterium indologenes PgBE177 of accession number KACC 92247P.
The composition for controlling Pseudomonas syringae ( Pseudomonas syringae ) comprising any one or more selected from the group consisting of the strain of claim 1, its culture medium and a mixture thereof.
Pseudomonas syringae ( Pseudomonas syringae ) control method comprising the step of treating the composition for control of claim 9.
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